Anandamide (AEA) and 2-arachidonylglycerol (2-AG) are the primary endogenous neurotransmitters in the endocannabinoid (EC) system. These chemical messengers, unlike the most well studied neurotransmitters, are lipids and, therefore, regulated through distinct biochemical processes. For the past decade it has been intensely debated whether the metabolic pathways responsible for EC synthesis and degradation are chiefly responsible for EC signal initiation and termination. Challenging this notion, evidence has recently emerged for the existence of specific EC-binding proteins that may facilitate EC uptake in neurons. The current model of 2- AG signaling has also been called into question based on several new studies suggesting that 2-AG is stored prior to release rather than being synthesized 'on-demand'. Direct evidence for specific EC-carrier proteins and for 2-AG storage is lacking, leaving these controversial hypotheses largely unsettled. To address these unresolved questions, we will attempt to refine the understanding of EC transport, storage, and release in the mammalian brain employing a multifaceted chemo-biological approach. First, we propose to comprehensively map EC-binding proteins in the nervous system by implementing activity-based protein profiling and multidimensional protein identification technologies (ABPP-MudPIT). EC-binding proteins will be enriched with the aid of tag-free photoaffinity probes and identified by multidimensional LC-MS/MS. Protein targets exhibiting high specificity and affinity will be further investigated in functional assays to elucidate their role in the EC signaling. Second, we propose to develop new methods to determine the cellular and subcellular localization of 2-AG in the mammalian brain. We will first attempt to develop 2-AG antibodies for immunofluorescence microscopy by synthesizing a terminally functionalized 2-AG analog enabling conjugation to an immunogenic carrier protein. We will also attempt to develop bioconjugation reactions that will allow fluorescent tagging of endogenous pools of 2-AG. Both strategies for imaging pools of 2-AG will be aided by the naturally high abundance of 2-AG relative to other unsaturated monoacylglycerol lipids and the selective increase of 2-AG levels in MAGL knockout mice. As a whole, this project has the potential to illuminate the mechanisms EC regulation and provide new therapeutic strategies for the treatment of EC-related disorders. PUBLIC HEALTH RELEVANCE: The endocannabinoids are a class of neurotransmitters that affect many normal physiological functions. The goal of this project is to investigate how endocannabinoids are regulated in the nervous system using chemical probes. This work has the potential to elucidate novel chemical strategies for investigating biological systems and to uncover novel therapeutic targets in the endocannabinoid system.